Liver failure is classified into several major types, including acute liver failure, chronic liver disease, and multiorgan failure. The main etiologies of liver failure are viral hepatitis and hepatotoxicity induced by drugs and toxins. Advanced liver failure results in encephalopathy and coma, and may be fatal. Treatment focuses on stabilization of the patient until spontaneous recovery of liver function, or until liver transplantation. In the aggregate, the annual mortality attributable to liver failure exceeds 27,000 annually in the United States.
Artificial organs, which are devices made entirely of non-biological materials, have greatly advanced health care. Artificial organs and tissue substitutes, including kidney dialysis machines, mechanical respirators, cardiac pacemakers, and mechanical heart pumps have sustained many people with desperate life-threatening diseases. The utility of such artificial organs is reflected in their widespread use. Pacemakers, for example, serve admirably as substitutes for their biological analogs.
The artificial kidney, sometimes termed the kidney dialysis machine, illustrates both the benefits and shortcomings of purely artificial organs. Kidney dialysis machines are effective in removing urea, creatinine, water, and excess salts from the blood, thus partly fulfilling major roles of the natural kidney. Clearly, artificial kidneys have postponed deaths of patients in renal failure. However, kidney dialysis machines are insufficiently selective and inappropriately remove biological components, such as steroid hormones, that a functioning natural kidney does not. Consequently, dialysis over an extended period may result in bone loss, clotting irregularities, immunodeficiencies, and sterility.
The patient in hepatic failure, unlike the patient in renal failure, cannot be specifically treated. Renal dialysis, which revolutionized the treatment of renal failure, does not presently have a hepatic equivalent. Currently, the only available treatment for refractory liver failure is hepatic transplantation. Many patients in hepatic failure do not qualify for transplantation because of concomitant infection, or other organ failure. Because of organ shortages and long waiting lists, even those who qualify for liver transplantation often die while awaiting an allograft. UCLA reported that one quarter of their transplant candidates died before a liver could be obtained. Organs suitable for transplant in the pediatric age group are even more scarce (Busuttil, R. W. et al. Ann Surg 1987, 206, 387).
The natural liver has four major classes of biochemical functions. First, the liver biosynthesizes a wide range of proteins, including major acellular components of blood, such as serum albumin, alpha-anti-trypsin, alpha-macroglobulin, enzymes, clotting factors, carrier molecules for trace elements, and the apo-lipoproteins. The liver then releases these components to the blood circulation. The liver also maintains appropriate plasma concentrations of amino and fatty acids. Second, the liver has a major role in detoxification reactions. The liver oxidizes or conjugates many harmful external poisons, processes that usually, but not always, diminish the poisonous character of the toxins. The liver also destroys excess hemoglobin, metabolizes the porphyrin molecules of hemoglobin, and recycles the iron component. Third, waste products, such as bilirubin, are conjugated and excreted via the biliary tree. Fourth, the liver synthesizes and secretes the bile salts, which serve as detergents that promote the emulsification and digestion of lipids. The multiplicity and biochemical character of liver function vastly increase the complexity of extracorporeal hepatic support.
Bioartificial organs are artificial organs designed to contain and sustain a viable biological component. Many biological functions are even more complex than simply generating a voltage potential at regular intervals, as occurs in the simplest of pacemakers. Examples include biosynthesis of blood components and catabolic processing of deleterious agents. The liver, endocrine glands, bone marrow, and kidney are prominent in such specialized biochemical functions. Artificial organs without a biological component cannot reproduce the complex biochemical functions executed by these organs.
Historically, non-biologic artificial liver substitutes have depended on hemodialysis and hemoperfusion, but have been of very short-term and highly limited benefit (Abe, T. et al., Therapeutic Apheresis 2000, 4:26). In contrast to purely artificial organs, an effective liver replacement must have a biological component. The liver is the most massive organ in the human body, exclusive of distributed organs such as skin, gut, hematopoietic system, and vasculature. Sustaining a large mass of functioning liver cells in vitro presents a variety of hurdles. At least eight major problems to developing a functional bioartificial liver can be described: 1) growing or obtaining appropriate and viable cells; 2) providing for a critical minimum mass of cells; 3) supplying oxygen to the cells; 4) supplying nutrients to the cells, and removing cell waste products efficiently; 5) limiting shear forces and hydrostatic pressures, 6) inducing or sustaining a differentiated cell phenotype with the capacity for biosynthesis and biotransformation of toxins; 7) maintaining sterility; and 8) preventing liver tissue rejection or lysis by complement.
1) Growing or obtaining appropriate and viable cells. Liver cells for potential use in bioartificial livers can be established cell lines, primary isolates from human or animal livers, or primordial liver cells however, secretion of tumorigenic factors is negatively affecting FDA approval of BAL designs incorporating cell lines (Xu, A. S. L. et al., 2000 in Lineage Biology and Liver, Lanza, R. P., Langer R., and Vacanti, J. (Ed.), Academic Press, San Diego, pp. 559–597). Cell lines of liver are available, for example HepG2 and C3A, that express many functions of differentiated liver. Cell lines offer the potential of growing sufficient numbers of cells in an extracorporeal mass cell culture system, or bioreactor, for sustaining a patient because the growth of cell lines is not limited by cell senescence, but by nutrient availability. Primary human or animal liver cells can also be obtained in the numbers required for a functional bioartificial liver. However, the use of human liver for cell preparation is limited by its lack of availability, and the use of animal liver for cell preparation suffers from some degree of cellular incompatibility. Acute cellular incompatibility results from the binding of antibodies that recognize foreign cells followed by the binding of proteins of the complement system and lysis of the foreign cells. Longer-term cellular incompatibility mechanisms also exist, but should not present any problems for the use of bioreactors as interim or “bridge” medical products. A possible alternative to initial inoculation with a large mass of differentiated cells is the expansion of liver stem cells that are progenitors of mature liver cells. Recent reports suggest that liver progenitor cells go through multiple cell divisions on the path toward maturation and differentiation (Brill, S. et al., Differentiation 1995, 59, 95; Sigal S. H. et al., Differentiation 1995, 59, 35). Suitable control of the growth and differentiation processes with staged application of appropriate cytokines can permit preparation of a clinically useful quantity of cells.
2) Providing for a critical minimum mass of cells. The adult human liver has a mass of about 1400–1600 grams, and features a considerable reserve, or redundant, capacity. It is estimated that human survival can be sustained with about 15–20% of the total liver mass. The figure of 20% of the liver mass corresponds to about 5×1010 cells (Kasai et al. Artif Organs 1994, 18, 348). Most, if not all, previous bioartificial liver designs suffer from a woefully inadequate cell capacity. That is, such devices are capable of sustaining far fewer than 5×1010 cells, often orders of magnitude fewer cells. Without the cell mass critical for biosynthesis of plasma components and detoxification reactions, these other designs have little clinical utility.
3) Supplying oxygen to the cells. The functional units of most organs such as nephron, acinus, alveoli, microvilli, skin, etc. consists of a capillary bed across which is a physico-chemical gradient. These gradients are controlled by mass transfer effects. Oxygen is the primary nutrient that is limiting in cell cultures (Macdonald, J. M. et al. NMR Biomed 1998, 11, 1; Glacken M. W. et al. Ann NY Acad Sci 1983, 413, 355). ‘Integral’ oxygenation, or aeration inside the bioreactor containing the biological or chemical material of interest, greatly enhances mass transfer of oxygen and carbonic acid. The formation of the latter can be used to control pH.
Oxygen is generally the limiting nutrient in hollow fiber bioartificial livers (Catapano, G. et al. Int J Art Organs 1996, 19, 61) primarily because hepatocytes are highly aerobic cells which causes problems of oxygen mass transfer. Oxygen has a relatively high diffusion coefficient and its mass transfer from blood in the liver sinusoids to hepatocytes is dominated by diffusion rather than convection (i.e., convection and perfusion are caused by pressure gradients). These effects are because an oxygen molecule is much smaller than other nutrients such as a glucose molecule, or than biosynthetic products such as proteins, and because the hepatocytes generate steep concentration gradients in bioartifical livers. With known rates of oxygen diffusion and oxygen consumption, and reasonable estimates of cell density, the diffusion distance at which oxygen utilization becomes the rate-limiting factor for growth is approximately 200 μm (Macdonald, J. M. et al., 1999, in Cell Encapsulation Technology and Therapeutics, Kuhtreiber, W., Lanza, R. P. and Chick, W. L. (Eds.) Birkhauser Boston, Cambridge, pp. 252–286. In bioartificial livers with serial oxygenation aerated with air, oxygen becomes axially limiting in perfusion media by 25 mm (Macdonald et al., 1999, supra).
Hepatocytes have a high metabolic rate and require a continuous oxygen supply. The oxygen consumption rate ranges from 0.59 to 0.7 nmole/s/106 cells for HepG2 cells (Smith, M. D. et al Int J Artif Organs 1996, 19, 36) and is 0.42 nmole/s/106 cells for isolated hepatocytes (Rotem, A. et al. Biotech Bioeng 1992, 40, 1286). Integral oxygenation, that is, continuous supply of oxygen along the path of media supply to the cells, is essential to supplying oxygen to liver cells. Serial oxygenation, which is oxygenation at one or a few places in the fluid line of media supply cannot sustain the mass of liver cells needed for an effective bioartificial liver. A difficulty with serial oxygenation is that the solubility of oxygen in aqueous media unsupplemented with oxygen carriers is so low that any oxygen present is quickly depleted by cell metabolism. In fact, in longitudinal flow along a conventional bioreactor semipermeable membrane, hepatocytes deplete oxygen within 2.5 centimeters along the path and therefore convective oxygen mass transfer via increasing Starling flow is improved. Increasing flow rates through conventional bioreactors can cause fiber breeches and adversely affect hepatocyte function (Callies, R. et al., Bio/Technology 1994 12:75). Thus, bioartificial liver designs that do not provide for adequate oxygen delivery are able to support only a limited number of cells. In addition, the flux of oxygen in a diffusion-limited system constrains cells to grow very near (less than about 0.2 mm) to the supply of oxygen. For example, U.S. Pat. No. 5,622,857 to Goffe discloses a bioreactor with some coaxial and some parallel semi-permeable hollow fibers. The Goffe design allows integral oxygenation but does not constrain the thickness of the cell compartment. The fiber-to-fiber spacing in that design is 3–5 mm so that there is not strict control of the oxygen diffusion distance. Similarly, U.S. Pat. No. 5,183,566 to Darnell et al. discloses a bioreactor with bundles of hollow fibers in parallel. The Darnell et al. design does not permit a multitude of individual multi-coaxial fiber bundles to be built-up with accurate and reproducible diffusion distances, and the design is not easily scaled-up. The Darnell et al. design uses bundles of parallel fibers, again not effectively addressing the issue of oxygen diffusion. Thus, a need remains for a bioreactor which deals effectively with the diffusion-limited thickness of the cell mass, with providing a critical mass of cells, and with supplying oxygen throughout the length of the bioreactor without adverse shear force effects.
4) Supplying nutrients to the cells, and removing cell waste products efficiently. The issue of supplying nutrients such as carbohydrates, lipids, minerals, and vitamins has been successfully solved by several variants of hollow fiber technology, and these features must be successfully incorporated into any viable bioartificial liver or bioartificial organ design. Similarly, the issue of removing metabolic wastes is usually handled by the same system that supplies the nutrients. The consumption rates for glutamate, pyruvate, and glucose are typically in the range of 0.03 to 0.3 nmol/s/106 cells, with reasonable assumptions for cell density and growth rate (Cremmer, T. et al. J Cell Physiol 1981, 106, 99; Imamura, T. et al. Anal Biochem 1982, 124, 353; Glacken, M. Dissertation 1987). The diffusion rates of oxygen in tissue are similar to those of pyruvate in water, and higher than those of glucose. As these consumption rates are less than the oxygen consumption rate, oxygen is the limiting nutrient in most conditions.
5) Limiting shear forces and hydrostatic pressure. For a given bioreactor there is an optimum balance of convection and diffusion for adequate oxygen mass transfer without creation of severe oxygen gradients. For example, using a nontoxic oxygen range, <0.4 mM (solubility constant is 1.06 mM/atm, for air solubility is 0.2 mM at 37° C.), the convective component of oxygen mass transfer should be increased as cells are increasingly farther than 0.2 mm from supply of oxygen (Macdonald et al., 1999, supra.). Although the partial oxygen tension in the liver sinusoid is about 70 mm Hg near the portal triad dropping to 20 mm Hg near the central vein, which equates to a range of 0.096 to 0.027 mM of free oxygen, the hemoglobin-bound oxygen ranges from 6.26 to 2.91 mM. The velocity of blood flow in the liver sinusoid is about 0.02 cm/s while the oxygen diffusion coefficient is about 4 orders-of-magnitude less, or 2×10−6 cm2/s. However, hepatic function is adversely affected with increasing shear forces, and in vivo hepatocytes are protected by a layer of endothelia and extracellular matrix in the space of Disse. Sufficient shear forces will kill hepatocytes. Others have found that shear forces induce specific cytochrome P450's (Mufti N. A. and Shuler, M. L., Biotechnol. Prog., 1995, 11, 659). A recent study has shown that liver regenerates faster with 90% than with 70% hepatectomy and this was attributed to greater shear forces (Sato, Y. et al., Surg. Today, 1997, 27, 518). However, this faster regeneration could also be due to enhanced oxygen, nutrient, and agonist mass transfer. Therefore, there is some maximum level of shear force that hepatocytes can sustain while still displaying optimal function. This maximum level can be increased if a layer of endothelia protects hepatocytes.
To increase convection, hydrostatic pressure gradients are increased. Elevated hydrostatic pressures can implode hepatocytes. Therefore, it is important to stay below these pressures. It is possible to cause 100% mortality of isolated rat hepatocytes by generating hydrostatic pressures of greater than 7 psi (>300 mm Hg) for longer than 2 minutes while inoculating these cells into coaxial bioreactor using a syringe.
6) Inducing or sustaining a differentiated cell phenotype with the capacity for biosynthesis and biotransformation of toxins. The use of the differentiated phenotype of liver cells is necessary to produce a useful bioartificial liver because the specialized functions of the liver, including biosynthesis of blood components and detoxification of toxins, are associated with the differentiated phenotype. These specialized functions are lost in whole, or in part, as the cells dedifferentiate, which often happens in isolated primary cell culture. In contrast, the form of liver cells capable of rapid growth is the dedifferentiated phenotype, leaving the practitioner to balance two opposing needs (Enat, R. et al. Proc Natl Acad Sci USA, 1984, 81, 1411). Some reports suggest that the phenotype of liver cells may be modulated by the presence of cytokines and extracellular matrix components. In particular, the extracellular matrix components rich in collagen IV and laminin, produced by the Engelbrech-Holm Sarcoma (EHS) cells and available commercially as MATRIGEL™, when used with hormonally defined media induces a differentiated phenotype (Enat, R. et al., supra; Bissell, D. M. Scan J Gasterenterol-Suppl 1988, 151,1; Brill, S. et al. Proc Soc Exp Biol Med 1993, 204, 261).
7) Maintaining sterility. The implementation of facile sterilization procedures for bioreactors and associated components is essential for clinical utility of extracorporeal bioartificial organs. Fortunately, the procedures for sterilization are well established, including standard methods both for sterilization of extracorporeal devices and for maintaining asepsis by standard in-line filters.
8) Preventing liver tissue rejection or lysis by complement. Rejection of foreign tissue may occur by a rapid process known as complement-mediated lysis that involves binding of circulating antibodies to the foreign cell surface, attachment of the proteins of the complement system, and lysis of the offending cell. The cell-mediated immune system is responsible for delayed rejection reactions. However, the cell-mediated immune system should not play a major role in bioreactor systems that do not permit direct contact of host and donor cells. Foreign body reactions, for example, against the structural components of bioreactors, are also cell-mediated and should therefore not constitute substantial obstacles.
Needed Improvements.
In view of the above, a clear need exists for bioartificial livers to sustain patients in liver failure. Specifically, a need exists for an improved version of a bioartificial liver that would have a high biological cell capacity in very thin layers of cells, readily accessible to oxygen and nutrients. There is a need for an apparatus or bioreactor, that provides efficient oxygen delivery to large masses of cells in cell culture and permits transfer of beneficial biosynthetic cell products to the patient. Similarly, there exists a need for effective methods of use of such an apparatus.
The problems with existing bioreactor designs include inadequate oxygenation, minimal capacity for the biological cell component, limited capability for removal of toxins, excessive shear and hydrostatic forces, and difficulty in transferring biosynthetic cell products for patient use. In addition, existing bioreactor designs have not dealt effectively with the diffusion-limited thickness of the cell mass, with providing a critical mass of cells, and with supplying oxygen throughout the length of the bioreactor.